16S rRNA Gene Sequence-Based Identification of Bacteria in Automatically Incubated Blood Culture Materials from Tropical Sub-Saharan Africa.

BACKGROUND: The quality of microbiological diagnostic procedures depends on pre-analytic conditions. We compared the results of 16S rRNA gene PCR and sequencing from automatically incubated blood culture materials from tropical Ghana with the results of cultural growth after automated incubation. METHODS: Real-time 16S rRNA gene PCR and subsequent sequencing were applied to 1500 retained blood culture samples of Ghanaian patients admitted to a hospital with an unknown febrile illness after enrichment by automated culture. RESULTS: Out of all 1500 samples, 191 were culture-positive and 98 isolates were considered etiologically relevant. Out of the 191 culture-positive samples, 16S rRNA gene PCR and sequencing led to concordant results in 65 cases at species level and an additional 62 cases at genus level. PCR was positive in further 360 out of 1309 culture-negative samples, sequencing results of which suggested etiologically relevant pathogen detections in 62 instances, detections of uncertain relevance in 50 instances, and DNA contamination due to sample preparation in 248 instances. In two instances, PCR failed to detect contaminants from the skin flora that were culturally detectable. Pre-analytical errors caused many Enterobacteriaceae to be missed by culture. CONCLUSIONS: Potentially correctable pre-analytical conditions and not the fastidious nature of the bacteria caused most of the discrepancies. Although 16S rRNA gene PCR and sequencing in addition to culture led to an increase in detections of presumably etiologically relevant blood culture pathogens, the application of this procedure to samples from the tropics was hampered by a high contamination rate. Careful interpretation of diagnostic results is required

Results of 917 bp 16S rRNA gene sequencing of culture-positive samples.

<p>^aComprising <i>Escherichia coli</i> (17), <i>Escherichia hermannii</i> (1), <i>Enterobacter cloacae</i> (1), <i>Klebsiella pneumoniae</i> (5), <i>Salmonella enterica</i> (11), <i>Salmonella</i> spp. (8), <i>Serratia</i> sp. (1) according to biochemical identification.</p><p>^bIncluding Gram-negative, not further identified coliform bacteria (3), Gram-negative, not further identified non-fermentative rod-shaped bacteria (1), <i>Sphingomonas paucimobilis</i> (2), <i>Streptococcus</i> Lancefield Group G (2), viridians group streptococci (1)</p><p>^cIncluding <i>Micrococcus</i> spp. (6), and <i>Rhizobium radiobacter</i> (1).</p><p>^d<i>Bacillus</i> sp. identified by PCR and sequencing.</p><p>^eBiochemically confirmed <i>Escherichia coli</i> not differentiable between <i>Escherichia coli</i> and <i>Shigella</i> spp. (4), biochemical result <i>Serratia</i> sp. vs. sequencing result <i>Enterobacter</i> sp. (1), biochemical result <i>Klebsiella pneumoniae</i> vs. sequencing result <i>Enterobacter</i> sp. (1).</p><p>^fBiochemically identification <i>Pseudomonas luteola</i> (2) vs. sequence-based identification as <i>Acinetobacter baumannii</i> (1) and <i>Acinetobacter</i> sp. (1), respectively.</p><p>^gCultural suspicion of Gram-negative, coliform bacteria (2) vs. sequence results <i>Bacillus</i> sp. (1) and <i>Microbacterium</i> sp. (1), biochemically identified <i>Sphingomonas paucimobilis</i> (2) vs. sequence results <i>Paenibacillus xylanilyticus</i> (1) and <i>Microbacterium oxydans</i> (1).</p><p>^hCultural suspicion of coagulase-negative staphylococci (5) vs. sequencing results <i>Bacterium</i> spp. (2), <i>Micrococcus luteus</i> (1), <i>Sphingomonas</i> sp. (1), and <i>Staphylococcus aureus</i> (1).</p><p>ⁱCultural suspicion of <i>Corynebacterium</i> spp. (3) vs. sequencing results <i>Arthrobacter</i> spp. (2) and <i>Microbacterium</i> sp. (1).</p><p>Results of 917 bp 16S rRNA gene sequencing of culture-positive samples.</p

Results of the 917 bp 16S rRNA gene PCR and the 357 bp 16S rRNA gene PCR from culture-negative samples.

<p>^aComprising <i>Escherichia coli</i> (4), <i>E</i>. <i>coli</i>/<i>Shigella</i> spp. (2), <i>Escherichia</i> sp. (1), <i>Enterobacter</i> spp. (9), <i>Ewingella americana</i> (2), <i>Ewingella</i> spp. (3), <i>Klebsiella pneumoniae</i> (1), <i>Salmonella enterica</i> (9), <i>Salmonella</i> spp. (2), <i>Serratia</i> spp. (5), <i>Yersinia</i> spp. (3).</p><p>^bComprising potential contaminations during sample acquisition, bacteria which might be associated with transient bacteremia, and sequences with too poor quality for identifications even on genus level, i.e., <i>Bacillus</i> sp. (1), <i>Micrococcus</i> sp. (1), <i>Mycobacterium</i> sp. (1), <i>Staphylococcus epidermidis</i> (2), <i>Staphylococcus</i> spp. (4), <i>Streptococcus gallolyticus</i> ssp. <i>pasteurianus</i> (1), non-interpretable results due to poor sequence quality (40).</p><p>^cComprising <i>Alicycliphilus</i> sp. (1), Alphaproteobacterium (4), <i>Bacillus halodurans</i> (1), <i>Bacterium</i> sp. (1), <i>Balneomonas</i> sp. (1), <i>Bradyrhizobium</i> spp. (5), <i>Caulobacter</i> spp. (26), <i>Cloacibacterium normanense</i> (2), cocci (not further defined) (1), <i>Diaphorobacter</i> spp. (155), Flexibacteriaceae (1), <i>Janibacter</i> sp. (1), <i>Luteimonas</i> sp. (1), multiply superimposing sequences (45), <i>Riemerella</i> sp. (1), <i>Sporichtya</i> sp. (1), <i>Thermonema</i> sp. (1).</p><p>^dIn addition to this one detection of <i>S</i>. <i>pneumoniae</i> DNA, <i>sodA</i> sequencing identified <i>S</i>. <i>pneumoniae</i> DNA in one additional culture-negative sample, in which 16S rRNA gene sequencing did not allow for an identification even on genus level due to poor sequence quality.</p><p>^eConfirmed by <i>sodA</i> sequencing.</p><p>Results of the 917 bp 16S rRNA gene PCR and the 357 bp 16S rRNA gene PCR from culture-negative samples.</p

Summarized results of cultural growth and 16S rRNA gene PCR.

<p>Summarized results of cultural growth and 16S rRNA gene PCR.</p

Results of 917 bp 16S rRNA gene sequencing and consecutive 357 bp 16S rRNA gene sequencing in case of negative 917 bp 16S rRNA gene PCR results of culture-positive samples.

<p>^aComprising <i>Escherichia coli</i> (17), <i>Escherichia hermannii</i> (1), <i>Enterobacter cloacae</i> (1), <i>Klebsiella pneumoniae</i> (5), <i>Salmonella enterica</i> (11), <i>Salmonella</i> spp. (8), <i>Serratia</i> sp. (1) according to biochemical identification.</p><p>^bIncluding Gram-negative, not further identified coliform bacteria (3), Gram-negative, not further identified non-fermentative rod-shaped bacteria (1), <i>Sphingomonas paucimobilis</i> (2), <i>Streptococcus</i> Lancefield Group G (2), viridians group streptococci (1).</p><p>^cIncluding <i>Micrococcus</i> sp. (6), and <i>Rhizobium radiobacter</i> (1).</p><p>^d<i>Bacillus</i> sp. identified by PCR and sequencing.</p><p>^eBiochemically confirmed <i>Escherichia coli</i> not differentiable between <i>Escherichia coli</i> and <i>Shigella</i> sp. (4) based on 16S rRNA gene sequencing or identified as <i>Enterobacter</i> sp. (1), biochemical result <i>Serratia</i> sp. vs. sequencing result <i>Enterobacter</i> sp. (1), biochemical result <i>Klebsiella pneumoniae</i> vs. sequencing result <i>Enterobacter</i> sp. (1), biochemical result <i>Salmonella</i> sp. vs. sequencing result <i>Diaphorobacter</i> sp. (1).</p><p>^fBiochemically identification <i>Pseudomonas luteola</i> (2) vs. sequence-based identification as <i>Acinetobacter baumannii</i> (1) and <i>Acinetobacter</i> sp. (1), respectively.</p><p>^gCultural suspicion of Gram-negative, coliform bacteria (2) vs. sequence results <i>Bacillus</i> sp. (1) and <i>Microbacterium</i> sp. (1), cultural suspicion of Gram-negative, non-fermentative rod-shaped bacteria (1) vs. sequence result <i>Nesterenkonia</i> sp., biochemically identified <i>Sphingomonas paucimobilis</i> (2) vs. sequence results <i>Paenibacillus xylanilyticus</i> (1) and <i>Microbacterium oxydans</i> (1).</p><p>^hCultural suspicion of coagulase-negative staphylococci (13) vs. sequencing results <i>Bacterium</i> spp. (2), <i>Corynebacterium</i> sp. (1), <i>Diaphorobacter</i> spp. (3), <i>Haematobacter massiliensis</i> (1), <i>Kocuria</i> sp. (1), <i>Micrococcus luteus</i> (1), <i>Micrococcus</i> sp. (1), <i>Pseudomonas</i> sp. (1), <i>Sphingomonas</i> sp. (1), and <i>Staphylococcus aureus</i> (1).</p><p>ⁱCultural suspicion of <i>Corynebacterium</i> spp. (5) vs. sequencing results <i>Arthrobacter</i> spp. (2), <i>Diaphorobacter</i> spp. (2), <i>Massilia aurea</i> (1), and <i>Microbacterium</i> sp. (1).</p><p>^jCultural suspicion of <i>Micrococcus</i> spp. (3) vs. sequence results <i>Diaphorobacter</i> sp. (1), <i>Kocuria marina</i> (1), and <i>Staphylococcus</i> sp. (1).</p><p>Results of 917 bp 16S rRNA gene sequencing and consecutive 357 bp 16S rRNA gene sequencing in case of negative 917 bp 16S rRNA gene PCR results of culture-positive samples.</p